TY - INPR A1 - Schaefer, Natascha A1 - Janzen, Dieter A1 - Bakirci, Ezgi A1 - Hrynevich, Andrei A1 - Dalton, Paul D. A1 - Villmann, Carmen T1 - 3D Electrophysiological Measurements on Cells Embedded within Fiber-Reinforced Matrigel T2 - Advanced Healthcare Materials N2 - 2D electrophysiology is often used to determine the electrical properties of neurons, while in the brain, neurons form extensive 3D networks. Thus, performing electrophysiology in a 3D environment provides a closer situation to the physiological condition and serves as a useful tool for various applications in the field of neuroscience. In this study, we established 3D electrophysiology within a fiber-reinforced matrix to enable fast readouts from transfected cells, which are often used as model systems for 2D electrophysiology. Using melt electrowriting (MEW) of scaffolds to reinforce Matrigel, we performed 3D electrophysiology on a glycine receptor-transfected Ltk-11 mouse fibroblast cell line. The glycine receptor is an inhibitory ion channel associated when mutated with impaired neuromotor behaviour. The average thickness of the MEW scaffold was 141.4 ± 5.7µm, using 9.7 ± 0.2µm diameter fibers, and square pore spacings of 100 µm, 200 µm and 400 µm. We demonstrate, for the first time, the electrophysiological characterization of glycine receptor-transfected cells with respect to agonist efficacy and potency in a 3D matrix. With the MEW scaffold reinforcement not interfering with the electrophysiology measurement, this approach can now be further adapted and developed for different kinds of neuronal cultures to study and understand pathological mechanisms under disease conditions. KW - 3D cultures Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244194 ER - TY - INPR A1 - Brenner, Marian A1 - Zink, Christoph A1 - Witzinger, Linda A1 - Keller, Angelika A1 - Hadamek, Kerstin A1 - Bothe, Sebastian A1 - Neuenschwander, Martin A1 - Villmann, Carmen A1 - von Kries, Jens Peter A1 - Schindelin, Hermann A1 - Jeanclos, Elisabeth A1 - Gohla, Antje T1 - 7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase T2 - eLife N2 - Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain. KW - 7,8-dihydroxyflavone (7,8-DHF) KW - pyridoxal phosphatase (PDXP) KW - vitamin B6 KW - PDXP inhibitors Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350446 ER - TY - JOUR A1 - Doppler, Kathrin A1 - Schuster, Yasmin A1 - Appeltshauser, Luise A1 - Biko, Lydia A1 - Villmann, Carmen A1 - Weishaupt, Andreas A1 - Werner, Christian A1 - Sommer, Claudia T1 - Anti-CNTN1 IgG3 induces acute conduction block and motor deficits in a passive transfer rat model JF - Journal of Neuroinflammation N2 - Background: Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown. Methods: In the present study, we aimed to model acute autoantibody exposure by intraneural injection of IgG of patients with anti-contacin-1 autoantibodies to Lewis rats. Patient IgG obtained during acute onset of disease (IgG3 predominant) and IgG from the chronic phase of disease (IgG4 predominant) were studied in comparison. Results: Conduction blocks were measured in rats injected with the “acute” IgG more often than after injection of “chronic” IgG (83.3% versus 35%) and proved to be reversible within a week after injection. Impaired nerve conduction was accompanied by motor deficits in rats after injection of the “acute” IgG but only minor structural changes of the nodes. Paranodal complement deposition was detected after injection of the “acute IgG”. We did not detect any inflammatory infiltrates, arguing against an inflammatory cascade as cause of damage to the nerve. We also did not observe dispersion of paranodal proteins or sodium channels to the juxtaparanodes as seen in patients after chronic exposure to anti-contactin-1. Conclusions: Our data suggest that anti-contactin-1 IgG3 induces an acute conduction block that is most probably mediated by autoantibody binding and subsequent complement deposition and may account for acute onset of disease in these patients. This supports the notion of anti-contactin-1-associated neuropathy as a paranodopathy with the nodes of Ranvier as the site of pathogenesis. KW - complement deposition KW - paranodopathy KW - anti-contactin-1 KW - CIDP KW - passive transfer KW - autoantibody Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200476 VL - 16 IS - 73 ER - TY - JOUR A1 - Appeltshauser, Luise A1 - Brunder, Anna-Michelle A1 - Heinius, Annika A1 - Körtvélyessy, Peter A1 - Wandinger, Klaus-Peter A1 - Junker, Ralf A1 - Villmann, Carmen A1 - Sommer, Claudia A1 - Leypoldt, Frank A1 - Doppler, Kathrin T1 - Antiparanodal antibodies and IgG subclasses in acute autoimmune neuropathy JF - Neurology: Neuroimmunology & Neuroinflammation N2 - Objective To determine whether IgG subclasses of antiparanodal autoantibodies are related to disease course and treatment response in acute- to subacute-onset neuropathies, we retrospectively screened 161 baseline serum/CSF samples and 66 follow-up serum/CSF samples. Methods We used ELISA and immunofluorescence assays to detect antiparanodal IgG and their subclasses and titers in serum/CSF of patients with Guillain-Barre syndrome (GBS), recurrent GBS (R-GBS), Miller-Fisher syndrome, and acute- to subacute-onset chronic inflammatory demyelinating polyradiculoneuropathy (A-CIDP). We evaluated clinical data retrospectively. Results We detected antiparanodal autoantibodies with a prevalence of 4.3% (7/161), more often in A-CIDP (4/23, 17.4%) compared with GBS (3/114, 2.6%). Longitudinal subclass analysis in the patients with GBS revealed IgG2/3 autoantibodies against Caspr-1 and against anti-contactin-1/Caspr-1, which disappeared at remission. At disease onset, patients with A-CIDP had IgG2/3 anti-Caspr-1 and anti-contactin-1/Caspr-1 or IgG4 anti-contactin-1 antibodies, IgG3 being associated with good response to IV immunoglobulins (IVIg). In the chronic phase of disease, IgG subclass of one patient with A-CIDP switched from IgG3 to IgG4. Conclusion Our data (1) confirm and extend previous observations that antiparanodal IgG2/3 but not IgG4 antibodies can occur in acute-onset neuropathies manifesting as monophasic GBS, (2) suggest association of IgG3 to a favorable response to IVIg, and (3) lend support to the hypothesis that in some patients, an IgG subclass switch from IgG3 to IgG4 may be the correlate of a secondary progressive or relapsing course following a GBS-like onset. KW - Guillain-Barre-Syndrome KW - inflammatory demyelinating polyradiculoneuropathy KW - musk myasthenia gravis KW - periperal nerve KW - neurofascin KW - autoantibodies KW - ontactin 1 KW - biopsies KW - binding KW - switch Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230079 VL - 7 IS - 5 ER - TY - JOUR A1 - Stengel, Helena A1 - Vural, Atay A1 - Brunder, Anna-Michelle A1 - Heinius, Annika A1 - Appeltshauser, Luise A1 - Fiebig, Bianca A1 - Giese, Florian A1 - Dresel, Christian A1 - Papagianni, Aikaterini A1 - Birklein, Frank A1 - Weis, Joachim A1 - Huchtemann, Tessa A1 - Schmidt, Christian A1 - Körtvelyessy, Peter A1 - Villmann, Carmen A1 - Meinl, Edgar A1 - Sommer, Claudia A1 - Leypoldt, Frank A1 - Doppler, Kathrin T1 - Anti–pan-neurofascin IgG3 as a marker of fulminant autoimmune neuropathy JF - Neurology: Neuroimmunology & Neuroinflammation N2 - Objective To identify and characterize patients with autoantibodies against different neurofascin (NF) isoforms. Methods Screening of a large cohort of patient sera for anti-NF autoantibodies by ELISA and further characterization by cell-based assays, epitope mapping, and complement binding assays. Results Two different clinical phenotypes became apparent in this study: The well-known clinical picture of subacute-onset severe sensorimotor neuropathy with tremor that is known to be associated with IgG4 autoantibodies against the paranodal isoform NF-155 was found in 2 patients. The second phenotype with a dramatic course of disease with tetraplegia and almost locked-in syndrome was associated with IgG3 autoantibodies against nodal and paranodal isoforms of NF in 3 patients. The epitope against which these autoantibodies were directed in this second phenotype was the common Ig domain found in all 3 NF isoforms. In contrast, anti–NF-155 IgG4 were directed against the NF-155–specific Fn3Fn4 domain. The description of a second phenotype of anti–NF-associated neuropathy is in line with some case reports of similar patients that were published in the last year. Conclusions Our results indicate that anti–pan-NF-associated neuropathy differs from anti–NF-155-associated neuropathy, and epitope and subclass play a major role in the pathogenesis and severity of anti–NF-associated neuropathy and should be determined to correctly classify patients, also in respect to possible differences in therapeutic response. KW - neurology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202462 VL - 6 IS - 5 ER - TY - JOUR A1 - Schaefer, Natascha A1 - Signoret-Genest, Jérémy A1 - von Collenberg, Cora R. A1 - Wachter, Britta A1 - Deckert, Jürgen A1 - Tovote, Philip A1 - Blum, Robert A1 - Villmann, Carmen T1 - Anxiety and Startle Phenotypes in Glrb Spastic and Glra1 Spasmodic Mouse Mutants JF - Frontiers in Molecular Neuroscience N2 - A GWAS study recently demonstrated single nucleotide polymorphisms (SNPs) in the human GLRB gene of individuals with a prevalence for agoraphobia. GLRB encodes the glycine receptor (GlyRs) β subunit. The identified SNPs are localized within the gene flanking regions (3′ and 5′ UTRs) and intronic regions. It was suggested that these nucleotide polymorphisms modify GlyRs expression and phenotypic behavior in humans contributing to an anxiety phenotype as a mild form of hyperekplexia. Hyperekplexia is a human neuromotor disorder with massive startle phenotypes due to mutations in genes encoding GlyRs subunits. GLRA1 mutations have been more commonly observed than GLRB mutations. If an anxiety phenotype contributes to the hyperekplexia disease pattern has not been investigated yet. Here, we compared two mouse models harboring either a mutation in the murine Glra1 or Glrb gene with regard to anxiety and startle phenotypes. Homozygous spasmodic animals carrying a Glra1 point mutation (alanine 52 to serine) displayed abnormally enhanced startle responses. Moreover, spasmodic mice exhibited significant changes in fear-related behaviors (freezing, rearing and time spent on back) analyzed during the startle paradigm, even in a neutral context. Spastic mice exhibit reduced expression levels of the full-length GlyRs β subunit due to aberrant splicing of the Glrb gene. Heterozygous animals appear normal without an obvious behavioral phenotype and thus might reflect the human situation analyzed in the GWAS study on agoraphobia and startle. In contrast to spasmodic mice, heterozygous spastic animals revealed no startle phenotype in a neutral as well as a conditioning context. Other mechanisms such as a modulatory function of the GlyRs β subunit within glycinergic circuits in neuronal networks important for fear and fear-related behavior may exist. Possibly, in human additional changes in fear and fear-related circuits either due to gene-gene interactions e.g., with GLRA1 genes or epigenetic factors are necessary to create the agoraphobia and in particular the startle phenotype. KW - glycine receptor KW - spastic KW - fear KW - anxiety KW - startle reaction Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-210041 SN - 1662-5099 VL - 13 IS - 152 ER - TY - JOUR A1 - Doppler, Kathrin A1 - Appeltshauser, Luise A1 - Krämer, Heidrun H. A1 - King Man Ng, Judy A1 - Meinl, Edgar A1 - Villmann, Carmen A1 - Brophy, Peter A1 - Dib-Hajj, Sulayman D. A1 - Waxman, Stephen G. A1 - Weishaupt, Andreas A1 - Sommer, Claudia T1 - Contactin-1 and Neurofascin-155/-186 Are Not Targets of Auto-Antibodies in Multifocal Motor Neuropathy JF - PLoS One N2 - Multifocal motor neuropathy is an immune mediated disease presenting with multifocal muscle weakness and conduction block. IgM auto-antibodies against the ganglioside GM1 are detectable in about 50% of the patients. Auto-antibodies against the paranodal proteins contactin-1 and neurofascin-155 and the nodal protein neurofascin-186 have been detected in subgroups of patients with chronic inflammatory demyelinating polyneuropathy. Recently, auto-antibodies against neurofascin-186 and gliomedin were described in more than 60% of patients with multifocal motor neuropathy. In the current study, we aimed to validate this finding, using a combination of different assays for auto-antibody detection. In addition we intended to detect further auto-antibodies against paranodal proteins, specifically contactin-1 and neurofascin-155 in multifocal motor neuropathy patients’ sera. We analyzed sera of 33 patients with well-characterized multifocal motor neuropathy for IgM or IgG anti-contactin-1, anti-neurofascin-155 or -186 antibodies using enzyme-linked immunosorbent assay, binding assays with transfected human embryonic kidney 293 cells and murine teased fibers. We did not detect any IgM or IgG auto-antibodies against contactin-1, neurofascin-155 or -186 in any of our multifocal motor neuropathy patients. We conclude that auto-antibodies against contactin-1, neurofascin-155 and -186 do not play a relevant role in the pathogenesis in this cohort with multifocal motor neuropathy. KW - motor proteins KW - enzyme-linked immunoassays KW - binding analysis KW - neuropathy KW - nerve fibers KW - cell binding assay KW - antibodies KW - enzyme assays Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126156 VL - 10 IS - 7 ER - TY - JOUR A1 - Janzen, Dieter A1 - Bakirci, Ezgi A1 - Wieland, Annalena A1 - Martin, Corinna A1 - Dalton, Paul D. A1 - Villmann, Carmen T1 - Cortical Neurons form a Functional Neuronal Network in a 3D Printed Reinforced Matrix JF - Advanced Healthcare Materials N2 - Impairments in neuronal circuits underly multiple neurodevelopmental and neurodegenerative disorders. 3D cell culture models enhance the complexity of in vitro systems and provide a microenvironment closer to the native situation than with 2D cultures. Such novel model systems will allow the assessment of neuronal network formation and their dysfunction under disease conditions. Here, mouse cortical neurons are cultured from embryonic day E17 within in a fiber‐reinforced matrix. A soft Matrigel with a shear modulus of 31 ± 5.6 Pa is reinforced with scaffolds created by melt electrowriting, improving its mechanical properties and facilitating the handling. Cortical neurons display enhance cell viability and the neuronal network maturation in 3D, estimated by staining of dendrites and synapses over 21 days in vitro, is faster in 3D compared to 2D cultures. Using functional readouts with electrophysiological recordings, different firing patterns of action potentials are observed, which are absent in the presence of the sodium channel blocker, tetrodotoxin. Voltage‐gated sodium currents display a current–voltage relationship with a maximum peak current at −25 mV. With its high customizability in terms of scaffold reinforcement and soft matrix formulation, this approach represents a new tool to study neuronal networks in 3D under normal and, potentially, disease conditions. KW - 3D electrophysiology KW - 3D neuronal networks KW - cortical neurons KW - melt electrowriting Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215400 VL - 9 IS - 9 ER - TY - JOUR A1 - Hecker, Katharina A1 - Grüner, Julia A1 - Hartmannsberger, Beate A1 - Appeltshauser, Luise A1 - Villmann, Carmen A1 - Sommer, Claudia A1 - Doppler, Kathrin T1 - Different binding and pathogenic effect of neurofascin and contactin–1 autoantibodies in autoimmune nodopathies JF - Frontiers in Immunology N2 - Introduction IgG4 autoantibodies against paranodal proteins are known to induce acute-onset and often severe sensorimotor autoimmune neuropathies. How autoantibodies reach their antigens at the paranode in spite of the myelin barrier is still unclear. Methods We performed in vitro incubation experiments with patient sera on unfixed and unpermeabilized nerve fibers and in vivo intraneural and intrathecal passive transfer of patient IgG to rats, to explore the access of IgG autoantibodies directed against neurofascin-155 and contactin-1 to the paranodes and their pathogenic effect. Results We found that in vitro incubation resulted in weak paranodal binding of anti-contactin-1 autoantibodies whereas anti-neurofascin-155 autoantibodies bound to the nodes more than to the paranodes. After short-term intraneural injection, no nodal or paranodal binding was detectable when using anti-neurofascin-155 antibodies. After repeated intrathecal injections, nodal more than paranodal binding could be detected in animals treated with anti-neurofascin-155, accompanied by sensorimotor neuropathy. In contrast, no paranodal binding was visible in rats intrathecally injected with anti-contactin-1 antibodies, and animals remained unaffected. Conclusion These data support the notion of different pathogenic mechanisms of anti-neurofascin-155 and anti-contactin-1 autoantibodies and different accessibility of paranodal and nodal structures. KW - autoimmune nodopathy KW - IgG4 KW - neurofascin KW - contactin KW - node of ranvier KW - inflammatory neuropathy KW - passive transfer Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320395 VL - 14 ER - TY - JOUR A1 - Atak, Sinem A1 - Langlhofer, Georg A1 - Schaefer, Natascha A1 - Kessler, Denise A1 - Meiselbach, Heike A1 - Delto, Carolyn A1 - Schindelin, Hermann A1 - Villmann, Carmen T1 - Disturbances of ligand potency and enhanced degradation of the human glycine receptor at affected positions G160 and T162 originally identified in patients suffering from hyperekplexia JF - Frontiers in Molecular Neuroscience N2 - Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GIyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GIyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GIyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, 1162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof. KW - mutations KW - trafficking KW - domain KW - hyperekplexia KW - loop B KW - side chain properties KW - ligand potencies KW - Cys-loop receptor KW - glycine receptor KW - site KW - activation KW - binding KW - channel KW - mechanisms KW - dominant KW - startle Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144818 VL - 8 IS - 79 ER - TY - JOUR A1 - Drehmann, Paul A1 - Milanos, Sinem A1 - Schaefer, Natascha A1 - Kasaragod, Vikram Babu A1 - Herterich, Sarah A1 - Holzbach-Eberle, Ulrike A1 - Harvey, Robert J. A1 - Villmann, Carmen T1 - Dual role of dysfunctional Asc-1 transporter in distinct human pathologies, human startle disease, and developmental delay JF - eNeuro N2 - Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5. Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5′ and 3′ untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1\(^{G307R}\) from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1\(^{G307R}\) did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay. KW - Asc-1 transporter KW - candidate gene KW - glycine receptor KW - glycine uptake KW - human startle disease KW - NMDAR Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349947 VL - 10 IS - 11 ER - TY - JOUR A1 - Schaefer, Natascha A1 - Zheng, Fang A1 - van Brederode, Johannes A1 - Berger, Alexandra A1 - Leacock, Sophie A1 - Hirata, Hiromi A1 - Paige, Christopher J. A1 - Harvey, Robert J. A1 - Alzheimer, Christian A1 - Villmann, Carmen T1 - Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease JF - Frontiers in Molecular Neuroscience N2 - Mutations in GlyR α1 or β subunit genes in humans and rodents lead to severe startle disease characterized by rigidity, massive stiffness and excessive startle responses upon unexpected tactile or acoustic stimuli. The recently characterized startle disease mouse mutant shaky carries a missense mutation (Q177K) in the β8-β9 loop within the large extracellular N-terminal domain of the GlyR α1 subunit. This results in a disrupted hydrogen bond network around K177 and faster GlyR decay times. Symptoms in mice start at postnatal day 14 and increase until premature death of homozygous shaky mice around 4–6 weeks after birth. Here we investigate the in vivo functional effects of the Q177K mutation using behavioral analysis coupled to protein biochemistry and functional assays. Western blot analysis revealed GlyR α1 subunit expression in wild-type and shaky animals around postnatal day 7, a week before symptoms in mutant mice become obvious. Before 2 weeks of age, homozygous shaky mice appeared healthy and showed no changes in body weight. However, analysis of gait and hind-limb clasping revealed that motor coordination was already impaired. Motor coordination and the activity pattern at P28 improved significantly upon diazepam treatment, a pharmacotherapy used in human startle disease. To investigate whether functional deficits in glycinergic neurotransmission are present prior to phenotypic onset, we performed whole-cell recordings from hypoglossal motoneurons (HMs) in brain stem slices from wild-type and shaky mice at different postnatal stages. Shaky homozygotes showed a decline in mIPSC amplitude and frequency at P9-P13, progressing to significant reductions in mIPSC amplitude and decay time at P18-24 compared to wild-type littermates. Extrasynaptic GlyRs recorded by bath-application of glycine also revealed reduced current amplitudes in shaky mice compared to wild-type neurons, suggesting that presynaptic GlyR function is also impaired. Thus, a distinct, but behaviorally ineffective impairment of glycinergic synapses precedes the symptoms onset in shaky mice. These findings extend our current knowledge on startle disease in the shaky mouse model in that they demonstrate how the progression of GlyR dysfunction causes, with a delay of about 1 week, the appearance of disease symptoms. KW - glycine receptor KW - startle disease KW - β8-β9 loop KW - mouse model KW - fast decay Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-196056 SN - 1662-5099 VL - 11 IS - 167 ER - TY - JOUR A1 - Kuhlemann, Alexander A1 - Beliu, Gerti A1 - Janzen, Dieter A1 - Petrini, Enrica Maria A1 - Taban, Danush A1 - Helmerich, Dominic A. A1 - Doose, Sören A1 - Bruno, Martina A1 - Barberis, Andrea A1 - Villmann, Carmen A1 - Sauer, Markus A1 - Werner, Christian T1 - Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy JF - Frontiers in Synaptic Neuroscience N2 - Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft. KW - super-resolution microscopy (SRM) KW - click-chemistry KW - dSTORM KW - GABA-A receptor KW - genetic code expansion Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251035 SN - 1663-3563 VL - 13 ER - TY - JOUR A1 - Rauschenberger, Vera A1 - von Wardenburg, Niels A1 - Schaefer, Natascha A1 - Ogino, Kazutoyo A1 - Hirata, Hiromi A1 - Lillesaar, Christina A1 - Kluck, Christoph J. A1 - Meinck, Hans‐Michael A1 - Borrmann, Marc A1 - Weishaupt, Andreas A1 - Doppler, Kathrin A1 - Wickel, Jonathan A1 - Geis, Christian A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine Receptor Autoantibodies Impair Receptor Function and Induce Motor Dysfunction JF - Annals of Neurology N2 - Objective Impairment of glycinergic neurotransmission leads to complex movement and behavioral disorders. Patients harboring glycine receptor autoantibodies suffer from stiff‐person syndrome or its severe variant progressive encephalomyelitis with rigidity and myoclonus. Enhanced receptor internalization was proposed as the common molecular mechanism upon autoantibody binding. Although functional impairment of glycine receptors following autoantibody binding has recently been investigated, it is still incompletely understood. Methods A cell‐based assay was used for positive sample evaluation. Glycine receptor function was assessed by electrophysiological recordings and radioligand binding assays. The in vivo passive transfer of patient autoantibodies was done using the zebrafish animal model. Results Glycine receptor function as assessed by glycine dose–response curves showed significantly decreased glycine potency in the presence of patient sera. Upon binding of autoantibodies from 2 patients, a decreased fraction of desensitized receptors was observed, whereas closing of the ion channel remained fast. The glycine receptor N‐terminal residues \(^{29}\)A to \(^{62}\)G were mapped as a common epitope of glycine receptor autoantibodies. An in vivo transfer into the zebrafish animal model generated a phenotype with disturbed escape behavior accompanied by a reduced number of glycine receptor clusters in the spinal cord of affected animals. Interpretation Autoantibodies against the extracellular domain mediate alterations of glycine receptor physiology. Moreover, our in vivo data demonstrate that the autoantibodies are a direct cause of the disease, because the transfer of human glycine receptor autoantibodies to zebrafish larvae generated impaired escape behavior in the animal model compatible with abnormal startle response in stiff‐person syndrome or progressive encephalitis with rigidity and myoclonus patients. KW - glycine receptor autoantibodies KW - behavioral disorders KW - neurology Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216005 VL - 88 IS - 3 SP - 544 EP - 561 ER - TY - JOUR A1 - Rauschenberger, Vera A1 - Piro, Inken A1 - Kasaragod, Vikram Babu A1 - Hörlin, Verena A1 - Eckes, Anna-Lena A1 - Kluck, Christoph J. A1 - Schindelin, Hermann A1 - Meinck, Hans-Michael A1 - Wickel, Jonathan A1 - Geis, Christian A1 - Tüzün, Erdem A1 - Doppler, Kathrin A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation JF - Frontiers in Molecular Neuroscience N2 - Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor’s glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera. KW - glycine receptor KW - autoantibodies KW - glycosylation KW - extracellular domain KW - adsorption Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304206 VL - 16 ER - TY - JOUR A1 - Schaefer, Natscha A1 - Vogel, Nicolas A1 - Villmann, Carmen T1 - Glycine receptor mutants of the mouse: what are possible routes of inhibitory compensation? JF - Frontiers in Molecular Neuroscience N2 - Defects in glycinergic inhibition result in a complex neuromotor disorder in humans known as hyperekplexia (OMIM 149400) with similar phenotypes in rodents characterized by an exaggerated startle reflex and hypertonia. Analogous to genetic defects in humans single point mutations, microdeletions, or insertions in the Glra1 gene but also in the Glrb gene underlie the pathology in mice. The mutations either localized in the (spasmodic, oscillator, cincinnati, Nmf11) or the (spastic) subunit of the glycine receptor (GlyR) are much less tolerated in mice than in humans, leaving the question for the existence of different regulatory elements of the pathomechanisms in humans and rodents. In addition to the spontaneous mutations, new insights into understanding of the regulatory pathways in hyperekplexia or glycine encephalopathy arose from the constantly increasing number of knock-out as well as knock-in mutants of GlyRs. Over the last five years, various efforts using in vivo whole cell recordings provided a detailed analysis of the kinetic parameters underlying glycinergic dysfunction. Presynaptic compensation as well as postsynaptic compensatory mechanisms in these mice by other GlyR subunits or GABA(A) receptors, and the role of extra-synaptic GlyRs is still a matter of debate. A recent study on the mouse mutant oscillator displayed a novel aspect for compensation of functionality by complementation of receptor domains that fold independently. This review focuses on defects in glycinergic neurotransmission in mice discussed with the background of human hyperekplexia en route to strategies of compensation. KW - GlyRs KW - rescue KW - hyperekplexia KW - knockout mice KW - spontaneous mouse mutants KW - synaptic inhibition Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123839 VL - 5 IS - 98 ER - TY - JOUR A1 - Wiessler, Anna-Lena A1 - Talucci, Ivan A1 - Piro, Inken A1 - Seefried, Sabine A1 - Hörlin, Verena A1 - Baykan, Betül B. A1 - Tüzün, Erdem A1 - Schaefer, Natascha A1 - Maric, Hans M. A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor β–targeting autoantibodies contribute to the pathology of autoimmune diseases JF - Neurology: Neuroimmunology & Neuroinflammation N2 - Background and Objectives Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα subunit–binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyRβ subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRβ making it not unlikely that GlyRβ-specific autoantibody (aAb) exist and contribute to the disease pathology. Methods In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRβ. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRβ binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyRβ aAb binding were resolved by whole-cell patch-clamp recordings. Results Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRβ aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRβ colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRβ aAb from both patients to its target impair glycine efficacy. Discussion Our study establishes GlyRβ as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRβ impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRβ aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization. KW - autoantibody (aAb) KW - glycine receptor (GlyR) KW - stiff-person syndrome (SPS) KW - clinical neurology KW - movement disorders KW - progressive encephalitis with rigidity and myoclonus (PERM) Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349958 VL - 11 IS - 2 ER - TY - JOUR A1 - Schaefer, Natascha A1 - Roemer, Vera A1 - Janzen, Dieter A1 - Villmann, Carmen T1 - Impaired Glycine Receptor Trafficking in Neurological Diseases JF - Frontiers in Molecular Neuroscience N2 - Ionotropic glycine receptors (GlyRs) enable fast synaptic neurotransmission in the adult spinal cord and brainstem. The inhibitory GlyR is a transmembrane glycinegated chloride channel. The immature GlyR protein undergoes various processing steps, e.g., folding, assembly, and maturation while traveling from the endoplasmic reticulum to and through the Golgi apparatus, where post-translational modifications, e.g., glycosylation occur. The mature receptors are forward transported via microtubules to the cellular surface and inserted into neuronal membranes followed by synaptic clustering. The normal life cycle of a receptor protein includes further processes like internalization, recycling, and degradation. Defects in GlyR life cycle, e.g., impaired protein maturation and degradation have been demonstrated to underlie pathological mechanisms of various neurological diseases. The neurological disorder startle disease is caused by glycinergic dysfunction mainly due to missense mutations in genes encoding GlyR subunits (GLRA1 and GLRB). In vitro studies have shown that most recessive forms of startle disease are associated with impaired receptor biogenesis. Another neurological disease with a phenotype similar to startle disease is a special form of stiff-person syndrome (SPS), which is most probably due to the development of GlyR autoantibodies. Binding of GlyR autoantibodies leads to enhanced receptor internalization. Here we focus on the normal life cycle of GlyRs concentrating on assembly and maturation, receptor trafficking, post-synaptic integration and clustering, and GlyR internalization/recycling/degradation. Furthermore, this review highlights findings on impairment of these processes under disease conditions such as disturbed neuronal ER-Golgi trafficking as the major pathomechanism for recessive forms of human startle disease. In SPS, enhanced receptor internalization upon autoantibody binding to the GlyR has been shown to underlie the human pathology. In addition, we discuss how the existing mouse models of startle disease increased our current knowledge of GlyR trafficking routes and function. This review further illuminates receptor trafficking of GlyR variants originally identified in startle disease patients and explains changes in the life cycle of GlyRs in patients with SPS with respect to structural and functional consequences at the receptor level. KW - glycine receptor KW - startle disease KW - autoimmune antibodies KW - protein maturation KW - trafficking pathways Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227531 VL - 11 IS - 291 ER - TY - JOUR A1 - Milanos, Sinem A1 - Elsharif, Shaimaa A. A1 - Janzen, Dieter A1 - Buettner, Andrea A1 - Villmann, Carmen T1 - Metabolic Products of Linalool and Modulation of GABA\(_{A}\) Receptors JF - Frontiers in Chemistry N2 - Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory α1β2 GABA\(_{A}\) receptors in various expression systems. However, in plants or humans, i.e., following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at α1β2γ2 GABA\(_{A}\) receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC\(_{10-30}\) together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself. KW - Cys-loop receptor KW - GABA\(_{A}\) KW - receptor KW - linalool KW - linalyl acetate KW - oxygenation KW - patch-clamp Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170779 VL - 5 IS - 46 ER - TY - JOUR A1 - Piro, Inken A1 - Eckes, Anna-Lena A1 - Kasaragod, Vikram Babu A1 - Sommer, Claudia A1 - Harvey, Robert J. A1 - Schaefer, Natascha A1 - Villmann, Carmen T1 - Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease JF - Frontiers in Molecular Neuroscience N2 - Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC\(_{50}\) value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations. KW - glycine receptor KW - hyperekplexia KW - startle disease KW - gephyrin Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246676 SN - 1662-5099 VL - 14 ER -